Validating Novel HR Variants in Cats

The two HR frameshift variants, including an exon 3 c.1255_1256dupGT, and the exon 18 c.3389insGACA, were identified by the WGS analyses. These variants were validated in the WGS cat by Sanger sequencing (Supplementary Table S1) A mass spectroscopy assay was designed as previously described [32 (link)] to genotype the identified variants in pedigree A (Supplementary Figure S1) and the additional cats, using the Agena Bioscience iPLEX Gold Genotyping reagent set (Agena Bioscience Inc., San Diego, CA, USA) (Supplementary Table S2). Products were genotyped with the MassARRAY System with Nanodispenser RS1000 (Agena Bioscience Inc., San Diego, CA, USA).
Not all ascertained cats with similar hair coats had the WGS identified variants, therefore the coding regions of HR were Sanger sequenced in each additional founder cat (Supplementary Table S1). PCR and thermocycling conditions were conducted as previously described [43 (link)]. The variants for the cats in the pedigree B (Supplementary Figure S2) were also genotyped by Sanger sequencing.